Acetyl CoA Assay Kit (ab87546)

1.We recommend using ~2-5 X 10 ^ 6 cells for each manipulation.
2. Deproteinization is recommended for cells in culture. I have attached the protocol that the lab recommends for this step.

In principle, eliminating the quenching and conversion step will allow you to measure CoA alone. However, the kit was not produced nor validated for this purpose, so we cannot guarantee that it will work, nor do we have a CoA standard that has been validated with this method to provide with the kit.



Alternatively, I would recommend one of our CoA detection kits listed below so you don't have to optimize and piece together the assay yourself.



Colorimetric or Fluorometric detection (detection range; 2.5 µM - 250 µM)

https://www.abcam.com/coenzyme-a-detection-kit-ab102504.html



Fluorometric detection (detection range: 40 nM - 10µM)

https://www.abcam.com/coenzyme-a-detection-kit-fluorometric-green-ab138889.html

J’ai contacté le laboratoire concernant le kit ab87546 :

1.Nous recommandons d’utiliser ˜2-5 X 10^6 cellules pour chaque manip.

2. Il est recommandé de faire une déprotéinisation pour des cellules en culture. J’ai attaché le protocole que le laboratoire recommande pour cette étape.

3. Comme expliqué au téléphone, quand vous ajoutez le « CoA conversion » à vos échantillons, vous devez ajouter le mélange qui correspond à la gamme standard que vous utilisez. Par exemple, si vous pensez que vos échantillons sont faibles en acetyl CoA, utilisez la gamme et le mélange « CoA conversion » de 0-100 pmol.

4. Effectivement, il doit y avoir eu une inversion des tubes « Acetyl CoA Standard (1 µmol) (Lyophilised) » et « Acetyl CoA Enzyme Mix » et notre stock a maintenant été vérifié.

1. Yes it is very crucial to do the sample prep exactly as recommended as Acetyl CoA is very sensitive to degradation and you risk the chance of inefficient results if you make changes to the protocol.
2. The deproteinization approach, though not as sensitive as the sample prep, would still benefit from using PCA rather than the 10 kDa spin filters. But you can use the spin filters if you do not have the option of using PCA.

Thank you for contacting us.

I have had the opportunity to discuss the specificity of this kit with the lab as you have requested. I have found that in this particular assay the probe is specific to just the NADH.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for your interest in our acetyl CoA assay kit, ab87546.

The assay measures relative amounts of acetyl CoA in samples indirectly, compared to a standard included with the kit, by converting the acetyl CoA to CoA, and then using the resulting CoA to reduce NAD to NADH, which in turn reacts with the probe to generate a fluorescent signal .

The conversion enzyme is what converts the acetyl CoA to CoA. The substrate mix is what the CoA acts upon, to generate the NADH. The components of the substrate mix and the identity of the conversion enzyme are considered proprietary information.

I hope this helps. Please let me now if you have any other questions.

The sample can be stored at -80 degree centigrade once prepared. However, we recommend to use the sample immediately after preparation for maximum efficiency.

I can confirm that kit PicoProbe Acetyl CoA Assay Kit (ab87546) can be used with frozen mouse tissues. Please see the protocol booklet for more information regarding sample preparation.

1. Mitochonrial lysate can be assayed by ab87546.

2. For the amount sample to be added into each sample well, we recommended to perform a pilot experiment with a range of dilutions of the sample to determine the optimal concentration/dilution of sample which gives the best results in the linear range of the standard curve. Please note that the probe in this kit is a very sensitive probe (minimum detection level is 10 pmol of Acetyl CoA). You may start with 1 ul sample to 10ul sample per well.